Acute tubular necrosis is a common and an important cause of acute renal failure (ARF) with aminoglycoside antibiotics including gentamicin accounting or 10 to 15 percent of all cases of ARF. Despite extensive investigations, the cellular mechanisms ultimately important in causing tubular injury and leading to ARF remain poorly understood. Based on a large body of evidence indicating that reactive oxygen metabolites (ROM) are important mediators of tissue injury, my overall hypothesis is that ROM play an important role in the subcellular and cellular changes that ultimately result in ARF. My major objective is to delineate a role for ROM in gentamicin nephrotoxicity. Towards this objective, I plan to examine the role of ROM in the subcellular and cellular effects of gentamicin and also to examined the direct subcellular and cellular effects of enzymatically generated ROM. Additional studies are planned to examine the role of ROM in other models of ARF where sufficient background information is available to postulate a role for ROM. The specific aims of this proposal are the following: A. to examine the effect of gentamicin on the generation of hydroxyl radical by renal cortical mitochondria. B. to examine the role of ROM in gentamicin-induced mitochondrial swelling and C. gentamicin-induced renal tubular cell cytotoxicity. In addition, the direct effects of enzymatically generate ROM on mitochondrial swelling and renal tubular cell cytotoxicity will be examined. D. In our recent study we found that hydroxyl radical scavengers and iron chelators are protective in gentamicin- induced ARF. Since the major source of hydroxyl radical in vivo is the interaction between superoxide anion and hydrogen peroxide (via the iron catalyzed Haber-Weiss reaction), I propose to examine the effects of scavengers of superoxide anion and hydrogen peroxide on gentamicin nephrotoxicity. E. Since iron is critical in the generation of hydroxyl radical, I propose to examine the effect of gentamicin and enzymatically generated ROM on the mobilization of iron from mitochondria, ferritin- loaded mitochondria and proximal tubular homogenates. In in vivo studies, the effect of gentamicin therapy on the renal cortical total and non-heme iron, and the effect of iron deficiency or iron overload on gentamicin-induced ARF will be examined. F. Based on the demonstrated role of reduced glutathione (GSH) in the protection of cells against the effects of ROM, I propose to examine in vitro, the effect of increasing or decreasing GSH levels on gentamicin-induced ARF. G. I propose to examine the role of ROM in those experimental models of acute tubular necrosis where sufficient background information is available to postulate a role for ROM e.g. other aminoglycoside-induced (Tobramycin, etc.), endotoxin-induced, glycerol-induced, and uranyl nitrate-induced ARF.